Journal: Journal of Animal Science

Article Title: Klebsiella pneumoniae causes mammary gland damage via FNIP1-mediated mitochondrial dysfunction

doi: 10.1093/jas/skaf384

Figure Lengend Snippet: K. pneumoniae inhibited milk fat and protein synthesis in BMECs. (A–C) mRNA levels of TNF-α , IL-1β , and IL-6 were detected by RT-qPCR method in BMECs (mean ± SEM, n = 3). (D) DEGs heat maps. (E) Enrichment analysis of differentially expressed genes KEGG. (F) The protein levels of SREBP1 and β-casein were detected in BMECs. (G, H) Relative protein abundance of SREBP1 and β-casein were normalized to β-actin (mean ± SEM, n = 3). (I, J) mRNA levels of SREBP1 and β-casein were detected by RT-qPCR method in BMECs (mean ± SEM, n = 3). (K) Typical images of BODIPY 493/503 staining in BMECs. * P < 0.05; ** P < 0.01.

Article Snippet: The following primary antibodies were used: SREBP1 (14088-1-AP, Proteintech, Hubei, China), β-casein (HY-P81091, MCE, Shanghai, China), OPA1 (DF8587, Affinity, Jiangsu, China), MFN1 (13798-1-AP, Proteintech, Hubei, China), cytochrome c oxidase I (COX I) (DF8920, Affinity, Jiangsu, China), DRP1 (12957-1-AP, Proteintech, Hubei, China), FIS1 (10956-1-AP, Proteintech, Hubei, China), FNIP1 (28380-1-AP, Proteintech, Hubei, China), β-actin (AF7018, Affinity, Jiangsu, China).

Techniques: Quantitative RT-PCR, Quantitative Proteomics, Staining

Journal: Journal of Animal Science

Article Title: Klebsiella pneumoniae causes mammary gland damage via FNIP1-mediated mitochondrial dysfunction

doi: 10.1093/jas/skaf384

Figure Lengend Snippet: K. pneumoniae caused mitochondrial dysfunction in mammary gland and dyssynthesis of milk fat and protein. (A) Representative images of H&E staining. (B) The severity of mastitis was assessed by differences in histological score ( n = 6 cows per group) between the control and K. pneumoniae infection groups. (C–G) mRNA levels of TNF-α , IL-1β , IL-6, SREBP1 , and β-casein were detected by RT-qPCR method in mammary gland tissues (mean ± SEM, n = 6). (H) The protein levels of SREBP1, β-casein OPA1, MFN1, COX I, DRP1, and FIS1 were detected in mammary gland tissues. (I–O) Relative protein abundance of OPA1, MFN1, COX I, DRP1, FIS1, SREBP1, and β-casein were normalized to β-actin (mean ± SEM, n = 3). (P) Relative ATP levels (mean ± SEM, n = 6). * P < 0.05; ** P < 0.01.

Article Snippet: The following primary antibodies were used: SREBP1 (14088-1-AP, Proteintech, Hubei, China), β-casein (HY-P81091, MCE, Shanghai, China), OPA1 (DF8587, Affinity, Jiangsu, China), MFN1 (13798-1-AP, Proteintech, Hubei, China), cytochrome c oxidase I (COX I) (DF8920, Affinity, Jiangsu, China), DRP1 (12957-1-AP, Proteintech, Hubei, China), FIS1 (10956-1-AP, Proteintech, Hubei, China), FNIP1 (28380-1-AP, Proteintech, Hubei, China), β-actin (AF7018, Affinity, Jiangsu, China).

Techniques: Staining, Control, Infection, Quantitative RT-PCR, Quantitative Proteomics

Journal: Journal of Animal Science

Article Title: Klebsiella pneumoniae causes mammary gland damage via FNIP1-mediated mitochondrial dysfunction

doi: 10.1093/jas/skaf384

Figure Lengend Snippet: Mdivi-1 recovered K. pneumoniae -induced mitochondrial damage and dyssynthesis of milk fat and protein in BMECs. (A) BMECs were treated with K. pneumoniae and/or 10 μM Mdivi-1 to analyze the protein levels of OPA1, MFN1, COX I, DRP1, and FIS1. (B–F) Relative protein abundance of OPA1, MFN1, COX I, DRP1, and FIS1 were normalized to β-actin (mean ± SEM, n = 3). (G) Relative ATP levels (mean ± SEM, n = 3). (H) BMECs were treated with K. pneumoniae and/or 10 μM Mdivi-1 to analyze the protein levels of SREBP1 and β-casein. (I, J) Relative protein abundance of SREBP1 and β-casein were normalized to β-actin (mean ± SEM, n = 3). (K, L) BMECs were treated with K. pneumoniae and/or 10 μM Mdivi-1 to analyze mRNA levels of SREBP1 and β-casein (mean ± SEM, n = 3). (M) Typical images of BODIPY 493/503 staining in BMECs. (N–P) BMECs were treated with K. pneumoniae and/or 10 μM Mdivi-1 to analyze mRNA levels of TNF-α , IL-1β , and IL-6 (mean ± SEM, n = 3). (Q) Detection of the content of LDH (mean ± SEM, n = 3). * P < 0.05; ** P < 0.01.

Article Snippet: The following primary antibodies were used: SREBP1 (14088-1-AP, Proteintech, Hubei, China), β-casein (HY-P81091, MCE, Shanghai, China), OPA1 (DF8587, Affinity, Jiangsu, China), MFN1 (13798-1-AP, Proteintech, Hubei, China), cytochrome c oxidase I (COX I) (DF8920, Affinity, Jiangsu, China), DRP1 (12957-1-AP, Proteintech, Hubei, China), FIS1 (10956-1-AP, Proteintech, Hubei, China), FNIP1 (28380-1-AP, Proteintech, Hubei, China), β-actin (AF7018, Affinity, Jiangsu, China).

Techniques: Quantitative Proteomics, Staining

Journal: Journal of Animal Science

Article Title: Klebsiella pneumoniae causes mammary gland damage via FNIP1-mediated mitochondrial dysfunction

doi: 10.1093/jas/skaf384

Figure Lengend Snippet: FNIP1 silencing alleviated K. pneumoniae -induced milk fat and protein dyssynthesis. (A) After FNIP1 silence, BMECs were infected with K. pneumoniae for 6 h to analyze the protein levels of SREBP1 and β-casein. (B, C) Relative protein abundance of SREBP1 and β-casein were normalized to β-actin (mean ± SEM, n = 3). (D, E) After FNIP1 silence, mRNA levels of SREBP1 and β-casein were detected by RT-qPCR method in BMECs (mean ± SEM, n = 3). (F) Typical images of BODIPY 493/503 staining in BMECs. (G–I) After FNIP1 silence, mRNA levels of TNF-α , IL-1β , and IL-6 were detected by RT-qPCR method in BMECs (mean ± SEM, n = 3). * P < 0.05; ** P < 0.01.

Article Snippet: The following primary antibodies were used: SREBP1 (14088-1-AP, Proteintech, Hubei, China), β-casein (HY-P81091, MCE, Shanghai, China), OPA1 (DF8587, Affinity, Jiangsu, China), MFN1 (13798-1-AP, Proteintech, Hubei, China), cytochrome c oxidase I (COX I) (DF8920, Affinity, Jiangsu, China), DRP1 (12957-1-AP, Proteintech, Hubei, China), FIS1 (10956-1-AP, Proteintech, Hubei, China), FNIP1 (28380-1-AP, Proteintech, Hubei, China), β-actin (AF7018, Affinity, Jiangsu, China).

Techniques: Infection, Quantitative Proteomics, Quantitative RT-PCR, Staining

Journal: Advanced Science

Article Title: Proline‐Selective Electrochemiluminescence Detecting a Single Amino Acid Variation Between A1 and A2 β‐Casein Containing Milks

doi: 10.1002/advs.202411956

Figure Lengend Snippet: a) Milk can contain either or both variants of β‐casein. The Pro‐to‐His substitution for the A1 variant facilitates proteolysis and formation of a β‐casomorphin‐7 (BCM‐7) bioactive peptide. b) Putative electrochemiluminescence (ECL) mechanisms that show the electrical and optical signals are linked although the linkage across these two modalities is amino‐acid‐dependent. c) Device used to simultaneously measure electrical and optical signals.

Article Snippet: To measure the amount of A1‐ and A2‐ β‐casein, a bovine A1 β‐casein and a A2 β‐casein sandwich ELISA assays (Biosensis, Australia) were used.

Techniques: Variant Assay, Electrochemiluminescence

Journal: Advanced Science

Article Title: Proline‐Selective Electrochemiluminescence Detecting a Single Amino Acid Variation Between A1 and A2 β‐Casein Containing Milks

doi: 10.1002/advs.202411956

Figure Lengend Snippet: Proline has a unique ECL response compared to other amino acids. a) Time series plots of input ( E ) and outputs (electrical, Q = ∫ i d t ; and optical, ECL ) for 5‐cycle cyclic voltammogram (CV). Lys and His have different response‐patterns and quantitative features ( Q Tot (Total Charge) and AUC Tot (Total Area Under Curve)). b) Phase plane analysis shows: Lys has a small electrical response (comparable to the Ru(bpy) 3 2+ control); His has a strong electrical (i.e., oxidative) response; and Pro has an intermediate electrical response but a very strong optical response (ECL). c) Our relative ECL responses for 15 amino acids compares with previous measurements [ ^22a ] using Spearman's correlation coefficients (r). A gray overlay indicates 95% confidence band for the best‐fit linear regression line (dotted line). d) Cluster analysis shows that Pro forms its own group (silhouette coefficient = 0.57). e) Electrical and optical responses vary linearly with amino acid concentration in an amino acid‐dependent manner. f) Cross‐modal analysis for His and Pro that differ between A1 and A2 β ‐casein variants (the dotted lines represent the fitted linear regression lines). All data are shown as the mean or the mean with the error bar presenting ± standard deviation (N = 4).

Article Snippet: To measure the amount of A1‐ and A2‐ β‐casein, a bovine A1 β‐casein and a A2 β‐casein sandwich ELISA assays (Biosensis, Australia) were used.

Techniques: Control, Concentration Assay, Standard Deviation

Journal: Advanced Science

Article Title: Proline‐Selective Electrochemiluminescence Detecting a Single Amino Acid Variation Between A1 and A2 β‐Casein Containing Milks

doi: 10.1002/advs.202411956

Figure Lengend Snippet: ELISA test kits and ECL can distinguish the protein standards for the A1 and A2 β‐casein variants. a) Schematic of ELISA and ECL measurements. b) As expected, ELISA kit designed to detect the A1‐variant shows low response to the A2‐variant. c) As expected, ELISA kit designed to detect the A2‐variant shows low response to the A1‐variant. All ELISA data are shown as the mean with the error bar representing ± standard deviation (N = 4). d) Time series input‐output plots and phase plane plots for the A1‐ and A2‐β‐casein standards. e) The electrical response metric ( Q Tot ) cannot distinguish the A1‐ and A2‐β‐caseins. f) The optical response metric ( AUC Tot ) can distinguish the A1‐ and A2‐β‐caseins. All ECL data are shown as the mean with the error bar representing ± standard deviation (N = 3). All p values in bar graphs were calculated with the Kruskal–Wallis test and the statistical significance is defined as: * = p < 0.05, ns (not siginificant) = p > 0.05.

Article Snippet: To measure the amount of A1‐ and A2‐ β‐casein, a bovine A1 β‐casein and a A2 β‐casein sandwich ELISA assays (Biosensis, Australia) were used.

Techniques: Enzyme-linked Immunosorbent Assay, Variant Assay, Standard Deviation

Journal: Advanced Science

Article Title: Proline‐Selective Electrochemiluminescence Detecting a Single Amino Acid Variation Between A1 and A2 β‐Casein Containing Milks

doi: 10.1002/advs.202411956

Figure Lengend Snippet: Validation of the ECL method by comparison with immunoanalysis. a) Immunoassays with A1‐specific and A2‐specific ELISAs show that the A2 milk has only the A2 β‐casein variant while regular milk has a mixture of A1 and A2 variants. Each bar is shown as the mean with the error presenting standard deviation (N = 3 for each milk). b) The correlations between the ELISA and ECL method indicate that the optical metric ( AUC Tot ) and cross‐modal metric ( AUC Tot / Q Tot ) distinguish regular and A2 milks based on their levels of their β‐casein variants (the electrical metric, Q Tot , by itself cannot discriminate regular and A2 milks). Spearman's correlation coefficients (r) are indicated. All gray overlays indicate 95% confidence bands for the best‐fit linear regression line (dotted line).

Article Snippet: To measure the amount of A1‐ and A2‐ β‐casein, a bovine A1 β‐casein and a A2 β‐casein sandwich ELISA assays (Biosensis, Australia) were used.

Techniques: Biomarker Discovery, Comparison, Variant Assay, Standard Deviation, Enzyme-linked Immunosorbent Assay

Journal: Advanced Science

Article Title: Proline‐Selective Electrochemiluminescence Detecting a Single Amino Acid Variation Between A1 and A2 β‐Casein Containing Milks

doi: 10.1002/advs.202411956

Figure Lengend Snippet: Comparison of the ECL method with conventional methods. a) The selectivity of the ECL is compared to gold standard immunoassays: data and Cluster analysis show that both the ELISA and ECL methods can distinguish A2 from regular milks (Silhouette coefficient = 0.87 (immunoassay); 0.6 (ECL)). b) The generic nature of the ECL method is illustrated by the cross‐modal metric ( AUC Tot / Q Tot ): this metric decreases as the analysis becomes more challenging (from distinguishing amino acids, to distinguishing proline‐rich peptides and proteins, and samples in complex backgrounds) yet the ECL method can discern the A2 and regular milks. p values were calculated with the Kruskal–Wallis test.

Article Snippet: To measure the amount of A1‐ and A2‐ β‐casein, a bovine A1 β‐casein and a A2 β‐casein sandwich ELISA assays (Biosensis, Australia) were used.

Techniques: Comparison, Enzyme-linked Immunosorbent Assay

Journal: Toxins

Article Title: PGN and LTA from Staphylococcus aureus Induced Inflammation and Decreased Lactation through Regulating DNA Methylation and Histone H3 Acetylation in Bovine Mammary Epithelial Cells

doi: 10.3390/toxins12040238

Figure Lengend Snippet: RT-qPCR validation of the differentially expressed genes (DEGs) obtained by RNA sequencing (RNA-Seq). ( A ) The genes involved in inflammation. ( B ) The genes involved in casein synthesis. Data represent the mean and standard deviation ( n = 6), and the asterisk indicates statistical difference (* p < 0.05) between the indicated columns, based on one-way analysis of variance and Duncan’s range test. IL-1β , interleukin-1β; IL-6 , interleukin-6; IL-8 , interleukin-8; TNF-α , tumor necrosis factor-α; CXCL1 , chemokine (C-X-C motif) ligand 1; CXCL6 , chemokine (C-X-C motif) ligand 6; CSN1S1 , αS1-casein; CSN2 , β-casein; CSN3 , κ-casein; RT-qPCR, reverse transcription quantitative real-time polymerase chain reaction; CON, control group; PGN, peptidoglycan group; LTA, lipoteichoic acid group; MIX, PGN + LTA group.

Article Snippet: The primary antibodies included CSN1S1 (Bioss Antibodies, bs-10034R, Beijing, China), CSN2 (Bioss Antibodies, bs-10032R, Beijing, China), CSN3 (Bioss Antibodies, bs-10031R, Beijing, China), Histone H3 (17168-1-AP, Proteintech, Wuhan, China), Histone H4 (16047-1-AP, Proteintech, Wuhan, China), acetylated Histone H3 (Acetyl Lys9/14, Bioss Antibodies, bs-4316R, Beijing, China), acetylated Histone H4 (Acetyl K5, Bioss Antibodies, bs-10721R, Beijing, China), and GAPDH (60004-1-Ig, Proteintech, Wuhan, China).

Techniques: Quantitative RT-PCR, RNA Sequencing Assay, Standard Deviation, Real-time Polymerase Chain Reaction

Journal: Toxins

Article Title: PGN and LTA from Staphylococcus aureus Induced Inflammation and Decreased Lactation through Regulating DNA Methylation and Histone H3 Acetylation in Bovine Mammary Epithelial Cells

doi: 10.3390/toxins12040238

Figure Lengend Snippet: The protein expression of the three caseins (CSN1S1, CSN2, and CSN3). The total protein was isolated to examine casein expression by Western Blot analysis, and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was selected as a housekeeping protein. Quantitation of blots is representative of three independent experiments. Data represent the mean and standard deviation ( n = 6), and the asterisk indicates statistical difference (* p < 0.05) between the indicated columns, based on one-way analysis of variance and Duncan’s range test. CSN1S1, αS1-casein; CSN2, β-casein; CSN3, κ-casein; CON, control group; PGN, peptidoglycan group; LTA, lipoteichoic acid group; MIX, PGN + LTA group.

Article Snippet: The primary antibodies included CSN1S1 (Bioss Antibodies, bs-10034R, Beijing, China), CSN2 (Bioss Antibodies, bs-10032R, Beijing, China), CSN3 (Bioss Antibodies, bs-10031R, Beijing, China), Histone H3 (17168-1-AP, Proteintech, Wuhan, China), Histone H4 (16047-1-AP, Proteintech, Wuhan, China), acetylated Histone H3 (Acetyl Lys9/14, Bioss Antibodies, bs-4316R, Beijing, China), acetylated Histone H4 (Acetyl K5, Bioss Antibodies, bs-10721R, Beijing, China), and GAPDH (60004-1-Ig, Proteintech, Wuhan, China).

Techniques: Expressing, Isolation, Western Blot, Protein Quantitation, Standard Deviation